Neutrophil extracellular traps (NETs) are extracellular lattices of decondensed chromatin decorated with antimicrobial proteins extruded by polymorphonuclear leukocytes (PMNs, neutrophils) to trap and kill microbes. Although NETs aid in trapping bacteria and other pathogens, their presence also leads to inflammatory tissue damage. Indeed, NET formation contributes to the pathology of several inflammatory disorders including acute lung injury resulting from influenza or blood transfusions, sepsis, small vessel vasculitis, systemic inflammatory response syndrome (SIRS), and chronic autoimmune diseases like systemic lupus erythematosus. Furthermore, NET formation and, in particular, the release of NET-associated histones into the extracellular space directly induces both epithelial and endothelial cell death in culture.
An active cell death process distinct from necrosis and apoptosis, frequently termed “NETosis,” leads to formation of three dimensional lattices studded with granule enzymes and host defense peptides that bind to nuclear chromatin before extrusion from the neutrophil. Histones, which have antimicrobial activities, are also abundant in NETs. NET formation is conserved in many species.
Deficient NET formation is a mechanism of immunodeficiency and impaired human host defense. A variety of pathogens induce NET formation. In addition to Escherichia coli, Staphylococcus, Streptococcus, Yersinia, and other gram positive and negative bacteria, fungi, parasites, and viruses trigger generation of NETs by human PMNs. Lipopolysaccharides (LPS) also induce NETosis, suggesting that microbial toxins may broadly have this activity. Endogenous host mediators, including interleukin-8 (IL-8), platelet activating factor (PAF), and complement factor C5a induce NET formation directly or after “priming” by other mediators. LPS stimulated mouse platelets, activated human platelets, and platelets in a murine model of transfusion-related acute lung injury (TRALI) that involves LPS priming also trigger NET formation. H1N1-infected alveolar epithelial cells induce NET formation by murine neutrophils in vitro. Thus, multiple interactions between neutrophils and microbes, host cells, and/or host mediators signal NETosis and NET formation.
NETs are also major biologic instruments of extravascular microbial containment and killing in vitro and in vivo, thus limiting the spread of pathogens. Certain pathogens, however, express endonucleases that cleave the DNA lattice or inhibitors that block antimicrobial peptides; these mechanisms act as virulence factors that limit killing and provide mechanisms for bacterial escape.
Failed NET formation appears to be a previously-unrecognized innate immune deficit that results in severe infections. Patients with chronic granulomatous disease (CGD), who are deficient in reactive oxygen species (ROS) generation and acquire recurrent, often life threatening bacterial and fungal infections, also demonstrate a defect in NET formation. Gene therapy for this immune deficiency can restore NET formation and control refractory pulmonary aspergillosis in patients with CGD. This suggests that NETs can also demonstrate protective functions in cystic fibrosis and pneumonia.
While the intracellular signaling pathways that regulate NET formation by PMNs remain largely unknown, ROS generation is considered a key event. Studies in human HL-60 myeloid leukocytes and genetically-altered mice indicate that activity of peptidylarginine deiminase 4 (PAD 4), an enzyme responsible for chromatin decondensation, is also required.
Additionally, NET formation may require enzymatic activity of neutrophil elastase (NE) to initiate degradation of core histones leading to chromatin decondensation prior to plasma membrane rupture. Alpha 1 anti-trypsin (A1AT) is a serine protease inhibitor that inactivates NE in plasma; it is, however, not expressed by human PMNs. A related serine protease inhibitor, serpin B1, which is expressed as a cytoplasmic protein by human PMNs, has been shown to restrict NET formation by mouse and human PMNs. Furthermore, treatment with recombinant serpin B1 inhibits NET formation in human PMNs stimulated with phorbol 12-myristate acetate (PMA), a robust inducer of NET formation in vitro. Recombinant A1AT, however, does not inhibit NET formation. Inhibition of specific serine proteases such as NE may effectively inhibit NET formation by human PMNs.
Although NET formation is a critical innate antimicrobial function of PMNs, there is now clear evidence that it is a mechanism of inflammatory tissue injury and thrombosis if inappropriately triggered and/or dysregulated. See Saffarzadeh and Preissner, Curr Opin Hematol, 2013, 20: 3-9; and Brinkmann and Zychlinsky, J Cell Biol, 2012, 198(5): 773-783. NETs mediate inflammatory damage in multiple models of sterile and infectious challenge. For example, NET formation may be a key mechanism in the systemic vasculopathy that is central to the pathogenesis of the acute, pro-inflammatory phase of sepsis. Bacteria including Staphylococci and E. coli are major causes of severe sepsis, alone or as polymicrobial infections, depending on the populations studied.
Experiments utilizing human endothelial cells (EC) and neutrophils in vitro, and an in vivo model of endotoxemia in which mice were challenged with LPS, indicate that NETs cause endothelial and liver damage, potentially mediated by neutrophil proteases associated with NETs. NE and other granule enzymes from neutrophils can potently injure endothelium and many extravascular cell types. EC activation, as occurs in sepsis, can enhance NET generation. In addition to granule enzymes, histones associated with NETs are previously-unrecognized agonists for endothelial injury in sepsis, based on experimental models and human samples.
There is also evidence that NETs and NET components are potent procoagulants, and that NET components induce thrombosis—a central pathogenetic feature of sepsis. NET components modify fibrin stability and fibrinolysis. Thus, while formation of NETs may be critical for bacterial capture and containment in the early phases of bacteremia and sepsis based on murine models, observations to date indicate that NET formation also causes damage to the host (for example, in acute septic syndromes).
Activated vascular endothelium may induce NET formation by human PMNs and lead to endothelial cell damage in vitro. Also, cellular damage may occur when human endothelial cells are incubated with activated platelets and PMNs, leading to NET formation, and liver injury may occur in vivo following NET formation. Finally, placentas from mothers with severe pre-eclampsia, a syndrome of pregnancy commonly leading to premature infant delivery, show exuberant NET formation. Thus, while essential in preventing severe infections, inappropriate NET formation appears to also be a mechanism of inflammatory vascular and tissue injury.
Inflammatory and infectious pulmonary syndromes provide another example of NET-mediated tissue injury. NETs form and contribute to vascular and alveolar dysfunction in animal models of acute lung injury and adult respiratory distress syndrome (ARDS). ARDS is a major complication of human systemic and pulmonary infection and inflammation. NETs are associated with acute lung injury in models of influenza-induced pneumonitis, a common and lethal infectious trigger for ARDS. In vitro studies indicate that NET histones may be critical mediators of alveolar endothelial and epithelial cell death.
In addition to sepsis and pulmonary injury, vasculitic syndromes are yet another example in which NETs play pathogenetic roles. As in sepsis, NETs may mediate both vascular inflammation and thrombosis in vasculitis. A variety of inflammatory stimuli and infectious agents can cause vasculitis.
Dysregulated inflammation has also been found to contribute to the pathogenesis of all the major complications of prematurity: necrotizing enterocolitis (NEC), respiratory distress syndrome (RDS), pneumonia, bronchopulmonary dysplasia (BPD), neonatal chronic lung disease (CLD), neurodevelopmental delay, retinopathy of prematurity (ROP), and sepsis. Neonatal CLD causes significant morbidity and mortality in the U.S. CLD is a complication of preterm birth that results from prolonged mechanical ventilation required for chronic respiratory failure. It occurs in up to 70% of mechanically ventilated extremely low birth weight infants (ELBW) with respiratory distress. While surfactant therapy and pre- or postnatal steroids have decreased the severity of CLD, this significant morbidity associated with preterm birth remains common in at-risk infants, with about 8,000 to 10,000 new cases occurring annually in the U.S. The mortality rate due to CLD in ELBW infants remains high from 15-60%. Furthermore, CLD remains the most common cause of long-term hospitalization in neonates and is also associated with developmental delay.
Additional current evidence strongly supports the conclusion that NETs are involved in tissue damage and thrombosis in a variety of inflammatory syndromes. Consequently, a few therapeutic strategies to blunt or interrupt NET formation or the activities of NET components have been investigated. All such strategies identified to date have major limitations, however. Global inhibition of neutrophil function and/or specific inhibitors of oxygen radical generation or key molecular checkpoints such as HIF-1α depress other PMN functions, including migration, phagocytosis, and/or intracellular microbial killing, causing parallel potential for microbial evasion and iatrogenic infections.
Disruption of NETs with DNases, potentially together with inhibition of NET-associated histones and enzymes as a combination strategy, represents a therapeutic approach based on experimental models. Nevertheless, more than twenty different NET-associated proteins have been identified, making this impractical. Furthermore, enzymatic disruption of NETs as an intervention may lead to dissemination of microbes, depending on its timing. Pharmacologic disruption of NETs in the vasculature also has the potential to spread histones and toxic neutrophil enzymes to other vascular beds, initiating or amplifying multiple organ injury. Finally, heparins inhibit NET formation under some conditions, but have bleeding as a well-known complication.